Summary: Rabbit Preimmune Repertoire Development We study genes of the rabbit immune system using techniques of molecular biology and immunology (1). In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs). We previously showed that diversification patterns in the clones from appendix were strikingly different from those found in splenic GCs where an immunizing antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual appendix clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign antigens. In collaboration with Dr. Ramit Mehr, we used a novel method for analyzing lineage tree shapes, using terms from graph theory to study diversification in rabbits and chickens. When lineage tree shapes were analyzed to quantify the differences between primary and secondary diversification in rabbits, the analyses indicated that primary diversification appears to occur at a constant rate in the appendix and the type of antigen-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut associated lymphoid tissues may play in early development of mammalian immune repertoires. The data also indicate a higher rate of hypermutation in rabbit during immune responses in splenic GCs, such that the balance between hypermutation and selection tends more towards mutation and less towards selection in rabbit compared to murine GCs (3). Microdissection of Single and Small numbers of Cells for DNA and RNA Analyses In order to collect single cells for PCR amplification and sequencing of rearranged VH genes, we have been using both the infra red based LCM and another UV laser-based microdissection system, Leica-LMD to collect appendix B lymphocytes. We also compared the tedious but successful method of manual hydraulic microdissection with techniques of laser capture microdissection (LCM). For these studies, we used both rabbit and human appendix tissues. Once capability to collect single cells by laser capture microdissection (LCM ) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR, products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for collection of single B cells (2). Both IR and UV lasers have been used for sample collection from tissue sections for genetic analyses. The high peak power densities in nitrogen laser microdissection (337nm) may excite endogenous photosensitizers or some histological dyes and cause DNA or RNA damage, perhaps through two photon mechanisms. We looked for diminished yields as evidence of damage when using LMD to isolate B cells. The Leica-LMD pulsed UV-laser system was used to collect single B cells from human appendix tissue sections, and single or multiple B cells from rabbit tissues, which had been immunohistochemically identified. Circles of different radii were used to assess possible loss of efficiency due to UV damage. The frequency of single allele PCR-amplification was compared between LMD and Hydraulic Micromanipulation (HM). Immunoglobulin VDJ PCR products and sequences obtained were indistinguishable for the two methods. We conclude that UV-based lasers can be used to cut cells individually from tissue sections provided the cutting edge is far enough from the cell membrane (>2.0 microns). In addition, with LMD we successfully isolated mRNA from rabbit splenic germinal centers (GC) containing antigen-specific B cells and determined gene sequences after RT-PCR and cloning. Total DNA and mRNA yields from 100-1000 cells collected in clusters were similar for LMD and HM. LMD appears particularly suited for independent collection of specific clusters of cells from anywhere on a slide and contiguous cell clusters from serial sections (2). Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. A collaboration with Dr. C. Rader and colleagues was established in order to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. Rabbits with the rare b9 and bas allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones is a rabbit/human Fab that binds with a dissociation constant of 1 nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer (5). Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Murine tumor angiogenesis models and receptor-specific antibodies are required to facilitate evaluation of the roles of VEGF receptors in mouse models of human cancer. We developed rabbit antibodies that cross-react with mouse (Flk-1) and human (KDR) VEGFR2. High-affinity, species cross-reactive, VEGFR2-specific Fabs were selected from an antibody phage display library generated from an immunized b9 allotype rabbit. The selected chimeric rabbit/human Fabs were found to bind to KDR and Flk-1 with nanomolar affinity. Three selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report further underscores our earlier finding that b9 allotype rabbits are excellent sources for generating high affinity cross-reactive antibodies with therapeutic potential (4). Methods: Rabbit immunization, RT PCR, cDNA library construction, PCR and genomic library construction, phage display library construction, immunohistochemistry, microdissection of single cells by hydraulic micromanipulation, laser capture microdissection (LCM) and Leica LMD, single-cell PCR and DNA sequencing, computer analyses of rearranged and germline Ig gene sequences, production of chimeric rabbit/human phage display libraries. Goals and Objectives: A long range objective of this research has been to define in molecular terms, the organization and regulated expression of rabbit immunoglobulin genes, describe their evolutionary origins, structures and molecular mechanisms leading to the diversification of variable regions. We use genetically defined rabbits in a variety of studies where knowledge of the molecular genetics and biology contributes to the conduct of the study. In addition, from time to time, we study other genes of importance in immune regulation and variable region diversification such as the rabbit recombination activating genes (RAG-1 and RAG-2), the RAD51 family of DNA repair genes and most recently rabbit activation induced deminase (AID).